Rapid Screening for Detecting Genetically Modified Wheat by Loop-Mediated Isothermal Amplification

We developed a loop-mediated isothermal amplification (LAMP)-based detection method for screening glyphosate-tolerant genetically modified (GM) wheat by targeting nucleotide sequences of the cauliflower mosaic virus 35S promoter (P35S) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Agrobacterium tumefaciens strain CP4. LAMP products were detected using a Genie II real-time fluorometer. The limit of detection (LOD) was found to be ≤0.05% corresponding to 14 copies of target DNA. We also designed primer sets for detecting the endogenous reference sequences of wheat, proline-rich protein (PRP), and Waxy-D1. The combination of two LAMP primer sets targeting the PRP and Waxy-D1 sequences was useful for distinguishing common wheat from durum wheat. Single-stranded tag hybridization (STH) on a chromatography printed-array strip (C-PAS) system, a lateral-flow DNA chromatography technology, was used to improve efficiency. LAMP amplification was clearly detected by the STH C-PAS system at the LOD level, and duplex detection of P35S and PRP, and EPSPS and PRP was successfully achieved. This simple and quick method for screening GM wheat will be useful for preventing food and environmental contamination by unauthorized GM crops.